As a medical diagnosis, pathological diagnosis is performed. In the pathological diagnosis, a pathologist diagnoses a disease using a tissue section collected from a human body and informs a clinician of whether or not a therapy and/or surgery is/are necessary. Based on the patient conditions and the pathological diagnosis, a physician determines the pharmacotherapeutic strategies and a surgeon determines whether or not a surgery should be performed.
In pathological diagnosis, it is common practice to prepare a tissue specimen by slicing a tissue sample obtained by evisceration or needle biopsy into a thickness of about several micrometers and then observe the tissue specimen at a magnification under a light microscope so as to obtain various findings. In many cases, a tissue specimen is prepared by fixing a collected tissue through dehydration and paraffin blocking, slicing the thus fixed tissue into a thickness of several micrometers and then removing the paraffin.
In pathological diagnosis, so-called immunostaining is performed for verifying the expression of an antigen or gene to be detected that is contained in a tissue specimen and then immunological observation is performed for diagnosing functional abnormalities such as abnormal expression of a gene or protein.
For immunostaining, for example, a dye staining method using an enzyme (e.g., DAB staining) is employed (Patent Document 1). In DAB staining, an antibody conjugated with peroxidase is used to stain an antigen, and the amount of the antigen is determined by adding thereto diaminobenzidine (DAB), which is the substrate of peroxidase, allowing it to show a color, and then observing the color.
However, in staining with an enzyme such as DAB staining, since the depth of color is largely variable depending on the environmental conditions such as temperature and time, there is a problem that estimation of the actual amount of an antigen or the like based on the depth of color is difficult.
Therefore, for immunological observation in pathological diagnosis, fluorescent labeling using a fluorescent label is performed as an alternative to staining with an enzyme label. This method characteristically has superior quantitative capability than DAB staining (Non-patent Document 1). In this method, the amount of the subject antigen is determined by staining the antigen with an antibody conjugated with a fluorescent dye and observing the stained antigen.
Since a tissue specimen hardly absorbs or scatters light and is thus nearly colorless and transparent, it is sometimes subjected to staining with a dye for morphological observation prior to being observed. There have been proposed a variety of staining techniques. In particular, for tissue specimens, hematoxylin-eosin staining (HE staining) using two dyes, hematoxylin and eosin, is typically used as a staining technique for observing the morphology of the subject tissue (Non-patent Document 1).
Hematoxylin stains cell nuclei, calcareous parts, cartilaginous tissues, bacteria and mucus in livid to light blue, while eosin stains cytoplasm, interstitial tissues, various fibers, erythrocytes and keratinocyte in red to dark red.
A pathologist makes a diagnosis based on the morphological and staining information, such as changes in the size and shape of cell nuclei and changes in the pattern as a tissue, in a micrograph of the stained tissue specimen. Examples of other staining for morphological observation include Papanicolaou staining (Pap staining) used for cytological diagnosis. By subjecting a tissue section to both morphological staining and immunostaining, morphological observation and immunological observation of the specimen also can be performed simultaneously.
In the preparation of a tissue specimen, aqueous mounting media and oil-based mounting media are known as mounting media for mounting a stained pathological section. Aqueous mounting media have a problem in that, since their refractive indices are generally largely different from that of a specimen, it is difficult to make a tissue specimen transparent. Meanwhile, oil-based mounting media are used for producing a permanent preparation not only because their refractive indices are not largely different from that of a tissue specimen and can thus make the tissue specimen transparent, but also because they allow good color tone and color development in morphological staining. Accordingly, oil-based mounting media are preferably used in the preparation of a tissue specimen.
As staining agents for tissue staining that are used in a fluorescent labeling method, staining agents which comprise resin particles containing a fluorescent substance immobilized thereon and which are used in a fluorescent dye labeling method (hereinafter, referred to as “dye-resin particles”) are known. In cases where such a staining agent is used for immunostaining of a tissue sample section and the thus stained tissue sample section is observed under a fluorescence microscope to quantify an antigen, the amount of adsorbed dye-resin particles can be evaluated based on the number of bright spots originating from the dye-resin particles.